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Erythroleukemia cells acquire an alternative mitophagy capability.

Wang J, Fang Y, Yan L, Yuan N, Zhang S, Xu L, Nie M, Zhang X, Wang J - Sci Rep (2016)

Bottom Line: Using CRISPR/Cas9 deletion of the canonical autophagy-essential gene Atg7, we found that erythroleukemia K562 cells are armed with two sets of autophagic machinery.This was accompanied by elevated ROS levels and apoptosis as well as reduced DNA damage repair.Therefore, the results suggest that erythroleukemia K562 cells possess an ATG7-independent alternative mitophagic mechanism that functions even when the canonical autophagic process is impaired, thereby maintaining the ability to respond to stresses such as excessive ROS and DNA damage.

View Article: PubMed Central - PubMed

Affiliation: Hematology Center of Cyrus Tang Medical Institute, Jiangsu Institute of Hematology, Collaborative Innovation Center of Hematology, Jiangsu Key Laboratory for Stem Cell Research, Soochow University School of Medicine, Suzhou 215123, China.

ABSTRACT
Leukemia cells are superior to hematopoietic cells with a normal differentiation potential in buffering cellular stresses, but the underlying mechanisms for this leukemic advantage are not fully understood. Using CRISPR/Cas9 deletion of the canonical autophagy-essential gene Atg7, we found that erythroleukemia K562 cells are armed with two sets of autophagic machinery. Alternative mitophagy is functional regardless of whether the canonical autophagic mechanism is intact or disrupted. Although canonical autophagy defects attenuated cell cycling, proliferation and differentiation potential, the leukemia cells retained their abilities for mitochondrial clearance and for maintaining low levels of reactive oxygen species (ROS) and apoptosis. Treatment with a specific inducer of mitophagy revealed that the canonical autophagy-defective erythroleukemia cells preserved a mitophagic response. Selective induction of mitophagy was associated with the upregulation and localization of RAB9A on the mitochondrial membrane in both wild-type and Atg7(-/-) leukemia cells. When the leukemia cells were treated with the alternative autophagy inhibitor brefeldin A or when the RAB9A was knocked down, this mitophagy was prohibited. This was accompanied by elevated ROS levels and apoptosis as well as reduced DNA damage repair. Therefore, the results suggest that erythroleukemia K562 cells possess an ATG7-independent alternative mitophagic mechanism that functions even when the canonical autophagic process is impaired, thereby maintaining the ability to respond to stresses such as excessive ROS and DNA damage.

No MeSH data available.


Related in: MedlinePlus

The alternative mitophagy is RAB9A-dependent.(A) Detection of the mRNA level of Rab9A by quantitative PCR in K562 cells. (B) Western blot analysis of RAB9A. (C) The colocalization of RAB9A and mitochondria in wild-type and Atg7−/− K562 cells treated with CCCP. The nuclei, RAB9A and mitochondria were respectively stained with DAPI (blue), Dylight 488 (green) and Deep Red (red). (D) The mitochondrial mass analysis in wild-type and Atg7−/− K562 ells treated with CCCP and Baf-A1 when Rab9A was knocked down. (E) The mitochondrial mass analysis in wild-type and Atg7−/− K562 cells treated with CCCP and BFA when Rab9A was knocked down. (F) Detection of the expression of mitochondrial protein TOMM20 by immunoblotting in wild-type and Atg7−/− K562 cells treated with CCCP and BFA when Rab9A was knocked down.
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f4: The alternative mitophagy is RAB9A-dependent.(A) Detection of the mRNA level of Rab9A by quantitative PCR in K562 cells. (B) Western blot analysis of RAB9A. (C) The colocalization of RAB9A and mitochondria in wild-type and Atg7−/− K562 cells treated with CCCP. The nuclei, RAB9A and mitochondria were respectively stained with DAPI (blue), Dylight 488 (green) and Deep Red (red). (D) The mitochondrial mass analysis in wild-type and Atg7−/− K562 ells treated with CCCP and Baf-A1 when Rab9A was knocked down. (E) The mitochondrial mass analysis in wild-type and Atg7−/− K562 cells treated with CCCP and BFA when Rab9A was knocked down. (F) Detection of the expression of mitochondrial protein TOMM20 by immunoblotting in wild-type and Atg7−/− K562 cells treated with CCCP and BFA when Rab9A was knocked down.

Mentions: As the upregulation of the alternative autophagy-essential protein RAB9A was associated with Atg7 deletion in K562 cells, we silenced Rab9A with RNA interference to analyze the importance of the RAB9A protein in alternative mitophagy in leukemia cells. Specifically, wild-type and Atg7−/− K562 cells were transfected with a lentivirus carrying shRNA against Rab9A. As shown by RT-PCR and western blotting results, Rab9A was successfully knocked down in both the wild-type and Atg7−/− K562 cells (Fig. 4A). Rab9A silencing was able to significantly suppress the CCCP-induced alternative degradation of mitochondria in both the wild-type and Atg7−/− K562, as observed in the mitochondria mass result obtained with flow cytometry (Fig. 4B). Confocal microscopy showed that RAB9A rarely colocalized with mitochondria under normal conditions, but CCCP treatment increased this colocalization in both the wild-type and Atg7−/− K562 cells (Fig. 4C, and Figure S1D), suggesting that CCCP-induced alternative mitophagy depends on RAB9A. As expected, when Rab9A was disrupted by RNA interference, CCCP was no longer able to induce the colocalization of RAB9A on mitochondria, suggesting a failure in the activation of alternative mitophagy (Fig. 4C). In support of this observation, the alternative autophagy inhibitor BFA also no longer inhibited mitochondrial autophagy, as determined by cytometric measurements of mitochondrial mass (Fig. 4D). Western blotting analysis also showed that when RAB9A was removed via gene silencing, the mitophagy marker TOMM20 did not change upon treatment with the mitophagy inducer CCCP (Fig. 4E), further suggesting that alternative mitophagy depends on RAB9A. TOMM20 is located in the membrane of mitochondria and thus can also be used for monitoring mitochondrial mass. Therefore, we exposed wild-type and Atg7−/− K562 leukemia cells to 3 Gy of nuclear radiation and observed the reduction of TOMM20 in both cell types in response to irradiation. The knockdown of RAB9A by small RNA interference inhibited the reduction of TOMM20 caused by the irradiation (Figure S1E).


Erythroleukemia cells acquire an alternative mitophagy capability.

Wang J, Fang Y, Yan L, Yuan N, Zhang S, Xu L, Nie M, Zhang X, Wang J - Sci Rep (2016)

The alternative mitophagy is RAB9A-dependent.(A) Detection of the mRNA level of Rab9A by quantitative PCR in K562 cells. (B) Western blot analysis of RAB9A. (C) The colocalization of RAB9A and mitochondria in wild-type and Atg7−/− K562 cells treated with CCCP. The nuclei, RAB9A and mitochondria were respectively stained with DAPI (blue), Dylight 488 (green) and Deep Red (red). (D) The mitochondrial mass analysis in wild-type and Atg7−/− K562 ells treated with CCCP and Baf-A1 when Rab9A was knocked down. (E) The mitochondrial mass analysis in wild-type and Atg7−/− K562 cells treated with CCCP and BFA when Rab9A was knocked down. (F) Detection of the expression of mitochondrial protein TOMM20 by immunoblotting in wild-type and Atg7−/− K562 cells treated with CCCP and BFA when Rab9A was knocked down.
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Related In: Results  -  Collection

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f4: The alternative mitophagy is RAB9A-dependent.(A) Detection of the mRNA level of Rab9A by quantitative PCR in K562 cells. (B) Western blot analysis of RAB9A. (C) The colocalization of RAB9A and mitochondria in wild-type and Atg7−/− K562 cells treated with CCCP. The nuclei, RAB9A and mitochondria were respectively stained with DAPI (blue), Dylight 488 (green) and Deep Red (red). (D) The mitochondrial mass analysis in wild-type and Atg7−/− K562 ells treated with CCCP and Baf-A1 when Rab9A was knocked down. (E) The mitochondrial mass analysis in wild-type and Atg7−/− K562 cells treated with CCCP and BFA when Rab9A was knocked down. (F) Detection of the expression of mitochondrial protein TOMM20 by immunoblotting in wild-type and Atg7−/− K562 cells treated with CCCP and BFA when Rab9A was knocked down.
Mentions: As the upregulation of the alternative autophagy-essential protein RAB9A was associated with Atg7 deletion in K562 cells, we silenced Rab9A with RNA interference to analyze the importance of the RAB9A protein in alternative mitophagy in leukemia cells. Specifically, wild-type and Atg7−/− K562 cells were transfected with a lentivirus carrying shRNA against Rab9A. As shown by RT-PCR and western blotting results, Rab9A was successfully knocked down in both the wild-type and Atg7−/− K562 cells (Fig. 4A). Rab9A silencing was able to significantly suppress the CCCP-induced alternative degradation of mitochondria in both the wild-type and Atg7−/− K562, as observed in the mitochondria mass result obtained with flow cytometry (Fig. 4B). Confocal microscopy showed that RAB9A rarely colocalized with mitochondria under normal conditions, but CCCP treatment increased this colocalization in both the wild-type and Atg7−/− K562 cells (Fig. 4C, and Figure S1D), suggesting that CCCP-induced alternative mitophagy depends on RAB9A. As expected, when Rab9A was disrupted by RNA interference, CCCP was no longer able to induce the colocalization of RAB9A on mitochondria, suggesting a failure in the activation of alternative mitophagy (Fig. 4C). In support of this observation, the alternative autophagy inhibitor BFA also no longer inhibited mitochondrial autophagy, as determined by cytometric measurements of mitochondrial mass (Fig. 4D). Western blotting analysis also showed that when RAB9A was removed via gene silencing, the mitophagy marker TOMM20 did not change upon treatment with the mitophagy inducer CCCP (Fig. 4E), further suggesting that alternative mitophagy depends on RAB9A. TOMM20 is located in the membrane of mitochondria and thus can also be used for monitoring mitochondrial mass. Therefore, we exposed wild-type and Atg7−/− K562 leukemia cells to 3 Gy of nuclear radiation and observed the reduction of TOMM20 in both cell types in response to irradiation. The knockdown of RAB9A by small RNA interference inhibited the reduction of TOMM20 caused by the irradiation (Figure S1E).

Bottom Line: Using CRISPR/Cas9 deletion of the canonical autophagy-essential gene Atg7, we found that erythroleukemia K562 cells are armed with two sets of autophagic machinery.This was accompanied by elevated ROS levels and apoptosis as well as reduced DNA damage repair.Therefore, the results suggest that erythroleukemia K562 cells possess an ATG7-independent alternative mitophagic mechanism that functions even when the canonical autophagic process is impaired, thereby maintaining the ability to respond to stresses such as excessive ROS and DNA damage.

View Article: PubMed Central - PubMed

Affiliation: Hematology Center of Cyrus Tang Medical Institute, Jiangsu Institute of Hematology, Collaborative Innovation Center of Hematology, Jiangsu Key Laboratory for Stem Cell Research, Soochow University School of Medicine, Suzhou 215123, China.

ABSTRACT
Leukemia cells are superior to hematopoietic cells with a normal differentiation potential in buffering cellular stresses, but the underlying mechanisms for this leukemic advantage are not fully understood. Using CRISPR/Cas9 deletion of the canonical autophagy-essential gene Atg7, we found that erythroleukemia K562 cells are armed with two sets of autophagic machinery. Alternative mitophagy is functional regardless of whether the canonical autophagic mechanism is intact or disrupted. Although canonical autophagy defects attenuated cell cycling, proliferation and differentiation potential, the leukemia cells retained their abilities for mitochondrial clearance and for maintaining low levels of reactive oxygen species (ROS) and apoptosis. Treatment with a specific inducer of mitophagy revealed that the canonical autophagy-defective erythroleukemia cells preserved a mitophagic response. Selective induction of mitophagy was associated with the upregulation and localization of RAB9A on the mitochondrial membrane in both wild-type and Atg7(-/-) leukemia cells. When the leukemia cells were treated with the alternative autophagy inhibitor brefeldin A or when the RAB9A was knocked down, this mitophagy was prohibited. This was accompanied by elevated ROS levels and apoptosis as well as reduced DNA damage repair. Therefore, the results suggest that erythroleukemia K562 cells possess an ATG7-independent alternative mitophagic mechanism that functions even when the canonical autophagic process is impaired, thereby maintaining the ability to respond to stresses such as excessive ROS and DNA damage.

No MeSH data available.


Related in: MedlinePlus